Rat Erythrocyte Membrane Research Articles

Age-related change in the alpha-tocopherolquinone/alpha-tocopherol ratio in the rat erythrocyte membrane

alpha-Tocopherol (alpha-Toc), a lipophilic phenolic antioxidant that is localized mainly in the biomembrane, protects cells against oxidation-associated cytotoxicity by prevention of membrane lipid peroxidation, maintenance of the redox balance intracellular thiols and stabilization of the membrane structure. We investigated the age-related changes in redox dynamics of alpha-Toc in plasma and erythrocyte membrane of an elderly (66 weeks old) and young group (10 weeks old). Total, alpha-, beta + gamma-, delta-Toc and alpha-tocopherolquinone (alpha-TocQ) in plasma and erythrocyte membrane were determined by high-performance liquid chromatography (HPLC) with a series of multiple coulometric working electrodes (CWE). Rat venous blood sample was divided into plasma and erythrocyte layers by centrifugation, and then erythrocyte membrane sample was prepared according to the method of Dodge et al. under a stream of nitrogen. In plasma, total and alpha-Toc concentrations were increased, and beta + gamma-, delta-Toc and alpha-TocQ concentrations were decreased age-dependently. In the erythrocyte membrane, total, alpha-TocQ concentrations and three fractions of tocopherols decreased age-dependently. Also, a decrease in the alpha-TocQ/alpha-Toc ratio in erythrocyte membrane was observed in the elderly group. These findings suggest that the alpha-Toc uptake in erythrocyte membrane and utilization rate of alpha-Toc in erythrocyte membrane decline age-dependently. This decline may promote membrane lipid peroxidation. alpha-Toc redox dynamics in erythrocyte membrane were useful to investigate the pathophysiology of aging mechanisms related to oxidative stress.

Alterations in rat erythrocyte membrane due to hexachlorocyclohexane (technical) exposure

Alterations in rat erythrocyte membrane due to hexachlorocyclohexane (technical) exposure

Alterations in rat erythrocyte membrane due to hexachlorocyclohexane (technical) exposure.

1. Hexachlorocyclohexane (HCH), an organochlorine pesticide having hydrophobic molecule is known to act on membranes. HCH mediated alterations in erythrocyte membrane occur through disorganization of the lipid bilayer. Therefore the changes in erythrocyte membrane fluidity, osmotic fragility and certain membrane bound enzymes were studied. Administration of HCH (technical) to rats at 5 mg/kg, orally, 5 days a week for 1, 2 and 3 months caused marked increase in erythrocyte membrane fluidity, osmotic fragility and decrease in levels of Na+, K(+)-ATPase, acetylcholinesterase in erythrocytes and glutathione in blood. 2. These changes indicate that HCH adversely affects membrane structure and function.

Adriamycin induces protein oxidation in erythrocyte membranes.

Adriamycin is an anthracycline antineoplastic agent whose clinical effectiveness is limited by severe side effects, including cardiotoxicity. A current hypothesis for adriamycin cardiotoxicity involves free radical oxidative stress. To investigate this hypothesis in a model system, we applied the technique of immunochemical detection of protein carbonyls, known to be increased in oxidized proteins, to study the effect of adriamycin on rat erythrocyte membranes. Erythrocytes obtained from adriamycin-treated rats demonstrated an increase of carbonyl formation in their membrane proteins. Yet, in separate experiments when adriamycin was incubated with rat erythrocyte ghosts, there was no significant increase of membrane protein carbonyls detected. In contrast, isolated erythrocytes incubated with an adriamycin-Fe3+ complex exhibited a robust carbonyl incorporation into their membrane proteins in a time-dependent manner. The level of carbonyl formation was dependent upon the concentration of Fe3+ known to form the adriamycin-Fe3+ complex. When the time course between protein carbonyl formation and lipid peroxidation was compared, protein carbonyl detection occurred earlier than lipid peroxidation as assayed by thiobarbituric acid reactive substances formation. These results are consistent with the notion that oxidative modification of membrane proteins may contribute to the development of the acute adriamycin-mediated toxicity.

Stability of rat erythrocyte membrane under chronic in vivo exposure to heat

Stability of rat erythrocyte membrane under chronic in vivo exposure to heat

Effect of n-3 fatty acid supplementation on lipid peroxidation and protein aggregation in rat erythrocyte membranes.

Human erythrocytes in the circulation undergo dynamic oxidative damage involving membrane lipid peroxidation and protein aggregation during aging. The present study was undertaken to determine the effect of n-3 fatty acid supplementation on lipid peroxidation and protein aggregation in the circulation and also the in vitro susceptibility of rat erythrocyte membranes to oxidative damage. Wistar male rats were fed a diet containing n-6 fatty acid-rich safflower oil or n-3 fatty acid-rich fish oil with an equal amount of vitamin E for 6 wk. n-3 Fatty acid content in erythrocyte membranes of rats fed fish oil was significantly higher than that of rats fed safflower oil. The degree of membrane lipid peroxidation and protein aggregation of rats fed fish oil was not significantly higher than that of rats fed safflower oil when the amounts of phospholipid hydroperoxides, thiobarbituric acid-reactive substances, and detergent-insoluble protein aggregates were measured. When isolated erythrocytes were oxidized under aerobic conditions in the presence of Fe(III), the degree of membrane lipid peroxidation of erythrocytes from rats fed fish oil was increased to a greater extent than that of rats fed safflower oil, whereas the degree of membrane protein aggregation of both groups was increased in a similar extent. Hence, n-3 fatty acid supplementation did not affect lipid peroxidation and protein aggregation in membranes of circulating rat erythrocytes, and the supplementation increased the susceptibility of isolated erythrocytes to lipid peroxidation, but not to protein aggregation, under the aerobic conditions. If a sufficient amount of vitamin E is supplied, n-3 fatty acid supplementation may give no undesirable oxidative effects on rat erythrocytes in the circulation.

Localization and quantification of the cytoskeleton-associated protein adducin in the kidneys of normal and Milan hypertensive rats.

Hypertension and kidney dysfunction in sodium transport observed in the Milan hypertensive strain (MHS) of rats are genetically associated with point mutations of adducin, an actin- and spectrin-binding protein of the membrane cytoskeleton. Polymorphism in the adducin locus has been reported to occur also in cases of human primary hypertension. In this study we show by immunostaining that adducin is localized along the basolateral epithelial membrane surface of the entire proximal and distal tubule with no detectable differences between MHS rats and the normotensive control strain (MNS). However, the total amount of adducin in kidney homogenates is reduced by about 45% in MHS rats as determined by quantitative immunoblotting. In erythrocyte membranes of MHS rats, adducin is reduced approximately 10%. The reduction of renal adducin in MHS rats is mainly caused by a reduction of the adducin pool that is loosely associated with kidney membranes and can be released by the non-ionic detergent, Triton X-100. The Triton-resistant, tightly membrane-bound pool of renal adducin differed by approximately 10% between MHS and MNS rats. Since several ion transporters have been shown to be tethered to the membrane cytoskeleton, we suppose that the reduction of the dynamic, loosely bound pool of adducin in MHS rats might interfere with the normal turnover and incorporation of yet unknown transporters involved in kidney sodium transport. However, the Na+,K+-ATPase appears to be not involved, as indicated by normal distribution and amounts of NA+,K+-ATPase in the kidney of MHS rats revealed by immunostaining and immunoblotting.

EFFECT OF NICKEL DEFICIENCY ON FATTY ACID COMPOSITION OF TOTAL LIPIDS AND INDIVIDUAL PHOSPHOLIPIDS IN BRAIN AND ERYTHROCYTES OF RATS

The present investigation was designed to examine the effect of nickel deficiency on fatty acid composition of total lipids and individual phospholipids in brain and erythrocyte membranes of rats. Therefore, a study over two generations was conducted feeding a nickel-deficient diet containing 13 μg/kg nickel or a nickel-adequate control diet supplemented with 1 mg/kg nickel. Male 7 week old rat pups from the second offspring were studied. The concentrations of individual brain phospholipids did not differ between the nickel-deficient and the nickel-adequate group, whereas in erythrocyte membranes the concentration of phosphatidylethanolamine (PE) diacyl was reduced in consequence of nickel depletion. Moreover, nickel deficiency slightly altered fatty acid composition of total lipids and individual phospholipids both in brain and erythrocytes. The changes in fatty acids were more marked in erythrocytes than in brain, and mainly concerned phosphatidylcholine (PC). The fatty acid composition in total lipids of brain was hardly affected by nickel. In brain individual phospholipids nickel deficiency slightly, but significantly elevated the proportions of polyunsaturated fatty acids (PUFA), and in the case of PC additionally increased the levels of monounsaturated fatty acids (MUFA) at the extent of saturated fatty acids (SFA). Total lipids of erythrocytes from nickel-deprived rats had a significantly higher proportion of total MUFA than nickel-adequate rats, and a somewhat lower proportion of total (n-6) PUFA. Individual phospholipids in erythrocytes from nickel-deficient pups tended to have lower PUFA levels than nickel-adequate rats, and had additionally inconsistently altered levels of SFA and MUFA in PC and PE plasmalogen, respectively. Moreover, individual erythrocyte phospholipids from nickel-deficient rats tended to have lower 20:418:2 ratios than nickel-adequate rats.Copyright © 1996 Elsevier Science Inc.

In vitro properties and organ uptake of rat band 3 cross-linked erythrocytes

Chemical conditions for cross-linking rat erythrocytes with bis(sulfosuccinimidyl)suberate (BS 3) and 3,3′ dithiobis(sulfosuccinimidyl propionate) (DTSSP) have been studied. These two cross-linking reagents seem to react with band 3 protein in rat erythrocyte membrane. Two different conditions have been assayed using different cell/cross-linker concentration ratios. Similar cell volumes were observed in cross-linked rat erythrocytes compared to rat control erythrocytes. Cell yields after cross-linking account for about 65% when a high ratio of cell-to-cross-linker was used. Slightly lower cell yields (about 62%) were obtained when this ratio was reduced. Estimation of band 3 cross-linking by gel electrophoresis revealed a level of cross-linking of around 45% and 50% at the different conditions used. In vivo behavior of these modified rat erythrocytes revealed that they do not circulate, showing a predominant localization in the liver. This effect is evident from the concentration (5 mM BS 3 or DTSSP) used. Based on these results, BS 3 and DTSSP can be considered as useful tools to cross-link rat erythrocyte band 3 and to target rat erythrocytes to the liver.

Spermine modulates calcium flux through the rat erythrocyte membrane.

The uptake of 45Ca by inside-out erythrocyte membrane vesicles (IOV) was dose-dependently inhibited by spermine. A cationic substance, Mg2+, did not affect this inhibitory activity of spermine. Ca(2+)-ATPase activity, however, was not inhibited by spermine at a concentration that could inhibit the uptake of 45Ca by the IOV. The release of 45Ca from IOV was significantly accelerated by spermine. Spermine bound to the surface of the IOV and was removed by treatment with acetic acid. These results suggest that spermine stimulates the release of Ca2+ from the IOV (or uptake of Ca2+ by erythrocytes) via an effect on the outer surface of the IOV (or the inner surface of erythrocytes).

In Vivo Oxidative Modification of Erythrocyte Membrane Proteins in Copper Deficiency

Oxidative stress has been postulated to contribute to the pathology associated with dietary copper deficiency. In vivo, erythrocytes are probable targets of oxidative damage because they are exposed to high concentrations of oxygen and contain heme iron that can autoxidize, which results in the formation of superoxide anions. Activity of the important antioxidant enzyme, copper, zinc superoxide dismutase, decreases markedly in erythrocytes during copper deficiency. The effect of dietary copper deficiency on indicators of oxidative stress was examined in erythrocyte membranes of rats maintained on a purified copper-deficient diet for 35 days after weaning. Erythrocytes were separated into young and old populations on a Percoll gradient prior to membrane isolation and quantification of lipid peroxides and protein carbonyls. Protein carbonyls, determined by Western blot immunoassay, were detected predominantly in both the alpha and beta chains of spectrin. Alpha and beta subunits of spectrin in erythrocyte membranes from copper-deficient rats contained higher amounts of carbonyls than controls, regardless of the population of erythrocytes studied. This study suggests that spectrin may be a specific target for oxidative damage when erythrocyte copper, zinc superoxide dismutase activity is reduced by copper deficiency. Copyright © 1997 Elsevier Science Inc.

Fatty Acid Composition of Erythrocyte Membranes Affects Iron Absorption in Rats

We studied the influence of the fatty acid composition of three different diets on iron absorption in relation to the lipid peroxidation of the erythrocyte membranes. After weaning, three groups of rats were fed isoenergetic semipurified diets for 16 wk: control diet (containing canola oil and peanut oil), tripalmitin diet (a saturated fatty acid diet) and a sardine oil diet rich in polyunsaturated fatty acids. Eight rats from each group were administered 59FeSO4 by gastric intubation for ferrokinetic studies, and six rats from each group were used for several analyses in blood, liver, spleen and erythrocytes. Results obtained from 59Fe eliminated in feces and 59Fe present in plasma, both calculated by the area under the curve and from maximum concentration in plasma, indicated that the sardine oil diet induced higher iron absorption than the control diet. The lipid peroxidation of erythrocyte membranes in rats fed sardine oil was higher than that in control rats due to high membrane's (n-3) polyunsaturated fatty acid content and low α-tocopherol level. The 59Fe in liver and spleen of rats fed the sardine oil diet was higher than in rats fed the control diet, and 59Fe was diverted to erythrocytes to a greater extent than in rats fed the control diet due to their reduced life-span. The sardine oil diet, by lessening α-tocopherol and raising the degree of lipid peroxidation in erythrocytes, augments iron absorption, whereas the tripalmitin diet, by protecting erythrocyte membranes from lipid peroxidation, leads to a lower rate of iron absorption.

3-Hydroxybutyrate decreases noradrenaline affinity for rat erythrocyte ghost beta 2-adrenergic receptors.

The effects of physiological levels of 3-hydroxybutyrate on noradrenaline binding to rat erythrocyte membranes were studied. The binding of noradrenaline to membranes was determined by measuring the union of 3H-noradrenaline in the presence of varying concentrations of 3-hydroxybutyrate. Scatchard plots of the results were used to determine that 3-hydroxybutyrate provoked a loss of affinity of the beta 2-adrenergic receptors for adrenaline. Thus high (but physiological) 3-hydroxybutyrate levels can lower the binding of the catecholamine, thus diminishing the extent or intensity of adrenergic-driven responses.

Effect of vitamin E on the lipid peroxidation induced by polyunsaturated fatty acids vis-a-vis cholesterol in rats fed Indian army ration

Effect of vitamin E on the lipid peroxidation induced by polyunsaturated fatty acids (PUFA) alone and PUFA in conjuction with cholesteril was stdied in liver and erythrocyte membrane of rats fed a natural mixed diet normally consumed by the Indian military personnel. Increased consumption of PUFA or PUFA together with cholesterol (eg. lacto-ovo-vegetarians) was found to have deleterious effect on the integrity of the tissue membranes, in vitro, due to peroxidative damage caused by the significant increase in the tissue lipid peroxidation rate. This effect was found to be more pronounced in the latter case. However, an increase in the vitamin E to PUFA ratio in the diet resulted in a notable counter effect.

Solubilization, reconstitution and molecular properties of the triiodothyronine transport protein from rat erythrocyte membranes.

Triiodothyronine (T3) transport through the mammalian erythrocyte membrane is mediated by a transport system related to the aromatic amino acid transport system T. The T3-binding component of this transport system could be photolabeled with [125I]T3 as a 52-kD protein, and subsequently solubilized with non-ionic detergents. Upon purification by ion-exchange chromatography, the photolabeled 52-kD protein solubilized with octylglucoside (OG) resolved into several peaks, suggesting charge heterogeneity of labeled proteins. The saturable [125I]T3 binding to rat erythrocyte membranes was completely inhibited by non-ionic detergents at concentrations about 20 times lower than those that solubilized membrane. Therefore, detergent-free proteoliposomes were generated from the detergent-soluble extracts by treatment with a polystyrene adsorbent. Proteoliposomes prepared from OG-soluble extract contained the highest specific activity of T3 binding. The Kd of the T3 binding sites (4.5 nmol/l) and the competitive inhibition constant of tryptophan (120 mumol/l) were similar to those for native membranes. The photolabeling of the 52-kD protein in these proteoliposomes was prevented by tryptophan and T4, but not by leucine or the D-isomer of T3, corresponding to the transport specificity of system T. The 52-kD protein solubilized with OG from native membranes was partially purified by ion-exchange chromatography. The 52-kD protein was detected by photoaffinity labeling in the purified fraction only after addition of erythrocyte membrane phospholipids to generate proteoliposomes. This indicates that the association of 52-kD protein with phospholipids is critical for T3 binding.

Simultaneous determination of α-tocopherol and α-tocopherolquinone by high-performance liquid chromatography and coulometric detection in the redox mode

A simple, selective and highly sensitive assay method for the simultaneous determination of α-tocopherol and α-tocopherolquinone in plasma or erythrocyte membrane by high-performance liquid chromatography (HPLC) with a series of multiple coulometric working electrodes (CWE) was investigated. For good separation of α-tocopherol and α-tocopherolquinone, an MC MEDICAL C 18 reversed-phase column and a mobile phase consisting of 96% methanol [methanol-HPLC-grade distilled water (96:4, v/v)] with 40 m M sodium perchlorate were used. Also, selective, highly sensitive and simultaneous detection of these substances was performed in redox mode using a series of four CWE. In this detection mode, the first, second and third CWE were set at −0.45 V for pre-reaction and to prevent interference, the fourth CWE was used as an electrode for actual measurement with its potential set at +0.40 V against a palladium reference electrode. The detection limits were 50–100 pg. Excellent chromatograms of α-tocopherol and α-tocopherolquinone were obtained within 8 min. The usefulness of reversed-phase HPLC with the redox detection mode was confirmed by application to the determination of the concentrations of α-tocopherol and α-tocopherolquinone in a crude ethanol-hexane extract of rat plasma or erythrocyte membrane. These findings suggest that reversed-phase HPLC with the redox detection mode using a series of four CWE is applicable to study the preventive effect of α-tocopherol on lipid peroxidation.

Abnormalities in Adenosine Triphosphatase of the Erythrocyte Membrane in Iron Deficiency Anaemia

During iron deficiency rat and human erythrocyte membrane enzyme activities (Total ATPase and Ouabainsensitive Na+.K+ ATPase) showed significant (P < 0.001) decrease. The influence of iron deficiency on erythrocyte Na+ and K+ was also studied in rats and humans. The former parameter showed a significant (P < 0.01) increase while the latter showed a downward trend. Plasma Fe and Total Iron Binding capacity (TIBC) in iron deficiency varied significantly (P < 0.05) from normal values. These results suggest a defect in erythrocyte membrane function and a possible potentiating effect of intracellular Na+, plasma Fe and TIBC on ATPase activity in iron deficiency. Values obtained for rats and humans showed differences in the activities of membrane ATPase in iron deficiency anaemia.

Effect of magnesium deficiency on fatty acid composition of the erythrocyte membrane and plasma lipid concentration in rats

We studied the effects of a magnesium (Mg)-deficient diet fed to rats (200 mg/kg of feed) on plasma lipid concentrations on days 7, 35, 42, 56, 63, and 70 and analyzed the changes in fatty acid composition of the erythrocyte membrane on day 70, Dietary Mg deficiency significantly increased free cholesterol, triglyceride, and phospholipid concentrations from day 7 of treatment and led to significant increases in total cholesterol from day 35 of treatment until the end of the experimental period. On day 70, Mg deficiency had reduced oleic acid (OA) (18:1n9) and arachidonic acid (AA) (20:4n6) in the erythrocyte membrane, and had increased eicosatrienoic (20:3n6), docosapentaenoic (22:5n6), and eicosapentaenoic acid (20:5n3). Our findings suggest that the decrease in AA after long-term (70 days) dietary Mg deficiency was not due to decreased Δ-6-desaturase activity, in contrast with earlier studies.

In vitro adsorption of amino acids onto isolated rat erythrocyte membranes

Amino acids adsorbed onto blood cell membranes represent about 8% of the total amino acids in blood. The aim of this study was to determine the in vitro adsorption kinetics of different amino acids (L-alanine, glycine, L-glutamate, L-glutamine, L-phenylalanine and L-leucine) onto rat erythrocyte membranes and to assess the effect of 24-hr starvation on these adsorption kinetics. Isolated red cell membranes were incubated at 37°C for 10 sec in the presence of 14C-amino acids—with different specific radioactivity—the radioactivity retained in the membrane fraction measured and kinetic parameters of amino acid adsorption determined. With the exception of glutamate, where the adsorption was negligible, all amino acids studied were adsorbed onto isolated red cell membranes, adhering to simple Michaelis-Menten kinetics. K m values of glycine, phenylalanine and leucine adsorption in control rats (14.7 ± 3.8 mM, 8.41 ± 0.95 mM and 4.65 ± 0.46 mM respectively, SEM, n = 6–8) decreased in response to 24-hr starvation, giving the following values: 0.792 ± 0.122 mM, 5.32 ± 0.82 mM and 3.53 ± 0.31 mM respectively (SEM, n = 6–8), V max, value of glycine adsorption of control rats decreased (from 61.0 ± 15.5 mmol/mol P/sec to 4.25 ± 0.70 mmol/mol P/sec, SEM, n = 7) and that of leucine increased (from 13.5 ± 1.0 mmol/mol P/sec to 18.9 ± 2.0 mmol/mol P/sec, SEM, n = 7) as an effect of 24-hr starvation. This study shows that alanine, glycine, glutamine, phenylalanine and leucine, but not glutamate, adsorbed onto erythrocyte membranes according to Michaelis-Menten-like kinetics. Furthermore, the adsorption was dependent on the nutritional status of the animal, which was consistent with a possible physiological role for the pool of amino acid bound to erythrocytes.

Insulin restores fatty acid composition earlier in liver microsomes than erythrocyte membranes in streptozotocin-induced diabetic rats

Alterations of fatty acid composition have been observed in a number of tissues in both experimental and human diabetes. Suppression of Δ6 desaturase in the liver, a key enzyme of fatty acid desaturation, has been reported to be responsible for these phenomena. We measured the fatty acid composition of the liver and the erythrocytes, and examined Δ6 desaturase activities to compare the effect of short-term insulin therapy on the tissues with and without Δ6 desaturase, ie., the liver and the erythrocytes using streptozotocin (STZ)-induced diabetic rats. Linoleic ( P < 0.05), palmitic ( P < 0.01) and docosahexaenoic (DHA) acid ( P < 0.01) were higher and arachidonic ( P < 0.01) and oleic acid ( P < 0.01) were lower in the liver microsomes of diabetic rats when compared to those in control rats. These alterations were partly reversed with insulin treatment. In the erythrocyte membrane, linoleic ( P < 0.01) and stearic acid ( P < 0.05) were higher, and palmitic ( P < 0.05), palmitoleic ( P < 0.01), and arachidonic acid ( P < 0.01) were lower in diabetic rats. In contrast to the case of the liver microsomes, however, these alterations were persistently observed after 48 h of insulin treatment. The activities of Δ6 desaturase in diabetic rats were 68% of those of controls ( P < 0.05), and increased to 119% of controls after insulin treatment. These results show that insulin restores the fatty acid composition earlier in the liver microsome than in the erythrocyte membrane in STZ-induced diabetic rats. The erythrocyte membrane would not be suitable for the investigation dealing with rapid changes of fatty acid composition.